Induction of intestinal stem cells by R-spondin 1 and Slit2 augments chemoradioprotection.

Cancer research has been rightly and successfully focused on prevention, early detection, and identification of specific molecular targets that distinguish the malignant cells from the neighbouring benign cells. However, reducing lethal tissue injury caused by intensive chemoradiotherapy during treatment of late-stage metastatic cancers remains a key clinical challenge. Here we tested whether the induction of adult stem cells could repair chemoradiation-induced tissue injury and prolong overall survival in mice. We found that intestinal stem cells (ISCs) expressed Slit2 and its single-span transmembrane cell-surface receptor roundabout 1 (Robo1). Partial genetic deletion of Robo1 decreased ISC numbers and caused villus hypotrophy, whereas a Slit2 transgene increased ISC numbers and triggered villus hypertrophy. During lethal dosages of chemoradiation, administering a short pulse of R-spondin 1 (Rspo1; a Wnt agonist) plus Slit2 reduced ISC loss, mitigated gut impairment and protected animals from death, without concomitantly decreasing tumour sensitivity to chemotherapy. Therefore Rspo1 and Slit2 may act as therapeutic adjuvants to enhance host tolerance to aggressive chemoradiotherapy for eradicating metastatic cancers.

Slit-Robo signaling induces malignant transformation through Hakai-mediated E-cadherin degradation during colorectal epithelial cell carcinogenesis.

The Slit family of guidance cues binds to Roundabout (Robo) receptors and modulates cell migration. We report here that ectopic expression of Slit2 and Robo1 or recombinant Slit2 treatment of Robo1-expressing colorectal epithelial carcinoma cells recruited an ubiquitin ligase Hakai for E-cadherin (E-cad) ubiquitination and lysosomal degradation, epithelial-mesenchymal transition (EMT), and tumor growth and liver metastasis, which were rescued by knockdown of Hakai. In contrast, knockdown of endogenous Robo1 or specific blockade of Slit2 binding to Robo1 prevented E-cad degradation and reversed EMT, resulting in diminished tumor growth and liver metastasis. Ectopic expression of Robo1 also triggered a malignant transformation in Slit2-positive human embryonic kidney 293 cells. Importantly, the expression of Slit2 and Robo1 was significantly associated with an increased metastatic risk and poorer overall survival in colorectal carcinoma patients. We conclude that engagement of Robo1 by Slit2 induces malignant transformation through Hakai-mediated E-cad ubiquitination and lysosomal degradation during colorectal epithelial cell carcinogenesis.

Slit2 regulates attractive eosinophil and repulsive neutrophil chemotaxis through differential srGAP1 expression during lung inflammation.

Directional migration of leukocytes is an essential step in leukocyte trafficking during inflammatory responses. However, the molecular mechanisms governing directional chemotaxis of leukocytes remain poorly understood. The Slit family of guidance cues has been implicated for inhibition of leuocyte migration. We report that Clara cells in the bronchial epithelium secreted Slit2, whereas eosinophils and neutrophils expressed its cell-surface receptor, Robo1. Compared to neutrophils, eosinophils exhibited a significantly lower level of Slit-Robo GTPase-activating protein 1 (srGAP1), leading to activation of Cdc42, recruitment of PI3K to Robo1, enhancment of eotaxin-induced eosinophil chemotaxis, and exaggeration of allergic airway inflammation. Notably, OVA sensitization elicited a Slit2 gradient at so-called bronchus-alveoli axis, with a higher level of Slit2 in the bronchial epithelium and a lower level in the alveolar tissue. Aerosol administration of rSlit2 accelerated eosinophil infiltration, whereas i.v. administered Slit2 reduced eosinophil deposition. In contrast, Slit2 inactivated Cdc42 and suppressed stromal cell-derived factor-1α-induced chemotaxis of neutrophils for inhibiting endotoxin-induced lung inflammation, which were reversed by blockade of srGAP1 binding to Robo1. These results indicate that the newly identified Slit2 gradient at the bronchus-alveoli axis induces attractive PI3K signaling in eosinophils and repulsive srGAP1 signaling in neutrophils through differential srGAP1 expression during lung inflammation.

Conversion of immortal liver progenitor cells into pancreatic endocrine progenitor cells by persistent expression of Pdx-1.

The conversion of expandable liver progenitor cells into pancreatic beta cells would provide a renewable cell source for diabetes cell therapy. Previously, we reported the establishment of liver epithelial progenitor cells (LEPCs). In this work, LEPCs were modified into EGFP/Pdx-1 LEPCs, cells with stable expression of both Pdx-1 and EGFP. Unlike previous work, with persistent expression of Pdx-1, EGFP/Pdx-1 LEPCs acquired the phenotype of pancreatic endocrine progenitor cells rather than giving rise to insulin-producing cells directly. EGFP/Pdx-1 LEPCs proliferated vigorously and expressed the crucial transcription factors involved in beta cell development, including Ngn3, NeuroD, Nkx2.2, Nkx6.1, Pax4, Pax6, Isl1, MafA and endogenous Pdx-1, but did not secrete insulin. When cultured in high glucose/low serum medium supplemented with cytokines, EGFP/Pdx-1 LEPCs stopped proliferating and gave rise to functional beta cells without any evidence of exocrine or other islet cell lineage differentiation. When transplanted into diabetic SCID mice, EGFP/Pdx-1 LEPCs ameliorated hyperglycemia by secreting insulin in a glucose regulated manner. Considering the limited availability of beta cells, we propose that our experiments will provide a framework for utilizing the immortal liver progenitor cells as a renewable cell source for the generation of functional pancreatic beta cells.

P-selectin primes leukocyte integrin activation during inflammation.

Selectins mediate leukocyte rolling and prime leukocytes for integrin-mediated leukocyte adhesion. However, neither the in vivo importance of nor the signaling pathway by which selectin-mediated integrin activation occurs has been determined. We report here that P-selectin-deficient mice manifested impaired leukocyte adhesion, which was 'rescued' by soluble P-selectin. Mechanistically, the cytoplasmic domain of P-selectin glycoprotein ligand 1 formed a constitutive complex with Nef-associated factor 1. After binding of P-selectin, Src kinases phosphorylated Nef-associated factor 1, which recruited the phosphoinositide-3-OH kinase p85-p110delta heterodimer and resulted in activation of leukocyte integrins. Inhibition of this signal-transduction pathway diminished the adhesion of leukocytes to capillary venules and suppressed peritoneal infiltration of leukocytes. Our data demonstrate the functional importance of this newly identified signaling pathway mediated by P-selectin glycoprotein ligand 1.

P-selectin cross-links PSGL-1 and enhances neutrophil adhesion to fibrinogen and ICAM-1 in a Src kinase-dependent, but GPCR-independent mechanism.

Endothelial and platelet P-selectin (CD62P) and leukocyte integrin alpha(M)beta(2) (CD11bCD18, Mac-1) are cell adhesion molecules essential for host defense and innate immunity. Upon inflammatory challenges, P-selectin binds to PSGL-1 (P-selectin glycoprotein ligand-1, CD162) to mediate neutrophil rolling, during which integrins become activated by extracellular stimuli for their firm adhesion in a G-protein coupled receptor (GPCR)-dependent mechanism. Here we show that cross-linking of PSGL-1 by dimeric or multimeric forms of platelet P-selectin, P-selectin receptor-globulin, anti-PSGL-1 mAb and its F(ab')2 induced adhesion of human neutrophils to fibrinogen (Fg) and intercellular cell adhesion molecule-1 (ICAM-1, CD54) and triggered a moderate clustering of alpha(M)beta(2), but monomeric forms of soluble P-selectin and anti-PSGL-1 Fab did not. Interestingly, P-selectin did not induce a detectable interleukine-8 (IL-8) secretion (<0.1 ng/ml) in 30 minutes, whereas a high concentration of IL-8 (>50 ng/ml) was required to increase neutrophil adhesion to Fg. P-selectin-induced neutrophil adhesion was significantly inhibited by PP2 (a Src kinase inhibitor), but not by pertussis toxin (PTX; a GPCR inhibitor). Activated platelets also increased neutrophil binding to fibrinogen and triggered tyrosine phosphorylation of cellular proteins. Our results indicate that P-selectin-induced integrin activation (Src kinase-dependent) is distinct from that elicited by cytokines, chemokines, chemoattractants (GPCR-dependent), suggesting that these two signal transduction pathways may cooperate for maximal activation of leukocyte integrins.

Naloxone acts as an antagonist of estrogen receptor activity in MCF-7 cells.

Estrogen promotes the growth of breast cancer via estrogen receptors (ER). Earlier studies showed that the opioid receptor antagonist naloxone inhibits MCF-7 breast cancer growth in mice. We examined the cellular and molecular mechanism of naloxone antagonism of ERalpha activity in human MCF-7 cells. Naloxone (100 nmol/L) inhibited 17beta-estradiol (E2)-induced (10 nmol/L) MCF-7 cell proliferation by 65% and mitogen-activated protein kinase/extracellular signal-regulated kinase phosphorylation. Naloxone blocked the E2-induced activation of ERalpha, with 85% inhibition after 5 minutes and 100% recovery after 60 minutes. This assay is based on quantitation of E2-activated nuclear ERalpha binding to the immobilized coactivator peptide. A significant decrease in E2-induced ERalpha transactivation was observed in the presence of naloxone in the estrogen response element-luciferase reporter assay (P < 0.05, E2 versus E2 + naloxone). Naloxone also blocked E2-induced down-regulation of ERalpha mRNA at 30 minutes and 6 hours. Although naloxone inhibits ERalpha activity directly, it also induces a cross-talk between mu-opioid receptor (MOR) and ERalpha. Immunoprecipitates with anti-MOR antibody showed the presence of ERalpha in cells incubated with E2 in the presence of naloxone but not in cells incubated with E2 or naloxone alone. Higher amounts of ERalpha associated with MOR after 60 minutes compared with 10 minutes of incubation. Naloxone inhibited E2-bovine serum albumin-FITC binding to plasma membrane-associated ERalpha and also inhibited the direct binding of [3H]E2 to ERalpha. Thus, naloxone modulates ERalpha activity directly as well as indirectly via MOR. This study suggests that naloxone-like compounds can be developed as novel therapeutic molecules for breast cancer therapy.